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NUCLEIC ACID SEQUENCING

The introduction of rapid methods of sequencing in the late ’70s represented a major shift in studies of two methods of DNA sequencing of nucleic acids. One of them (Maxam and Gilbert) chemical reagents used to cut DNA at specific base. The other is called enzymatic chain termination or dideoxy (Sanger, Nicklen and Coulson). The purpose of both is to get the whole sequence of each of the bases that form a nucleic acid fragment.

In the chemical method a DNA fragment is marked on one end with P or S radioactive by the enzyme polynucleotide kinase. Both ends are separated by the action of a restriction enzyme. The next step is to break these fragments, no more than once or twice per molecule, with specific chemical reactions for each of the four bases. We will discuss four aliquots and each of them with a chemical to alter one of the four bases, about once per molecule of DNA. Subsequent treatment with piperidine produce rupture of the chain where the base had been altered.

Fragments were subsequently analyzed in denaturing gels.

The enzymatic method is most used today. Prepare a linear circular DNA (cloned in phage M13). It introduces a primer that will serve for the initiation of DNA synthesis and we will make sequencing. The specific primer is complementary to a sequence of phage M13. A polymerase (Klenow enzyme or secuenasa) is responsible for curing. It sets four aliquots. The synthesis is carried out in two steps: the first part of the primer is extended a few hundred nucleotides utilizing the four nucleotides, but one of them is marked. In the second step occurs the termination of the chains by adding a small amount of a dideoxynucleotide different in each aliquot. The dideoxido is a nucleotide triphosphate that by lack groups OH 2’como both the 3 ‘carbon of ribose, thus preventing the growth of chain synthesis paralyzed. The result in each test tube is a collection of fragments of many sizes as there are units based on the sequence . The different fragments were analyzed by acrylamide gel electrophoresis.

Currently for marking using four different fluorochromes: fluorescein, NBD (4-chloro-7nitrobenceno-2oxal-Diazol) tetrametilrodamina and texas red, one for each base. . Once conjugated to four aliquots of the primer, we proceed to the extent of starting and stopping with dideoxis.